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The Inhibitory Effects of Green Tea (Camellia Sinensis) on the Growth and Proliferation of Oral Bacteria

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The Inhibitory Effects of Green Tea (Camellia Sinensis) on the Growth and Proliferation of Oral Bacteria THE INHIBITORY EFFECTS OF GREEN TEA (CAMELLIA SINENSIS) ON THE GROWTH AND PROLIFERATION OF ORAL BACTERIA Margaret Axelrod, Sean Berkowitz, Raina Dhir, Veronica Gould, Arjun Gupta, Eric Li, Jane Park, Amar Shah, Kevin Shi, Christelle Tan, Ming-Ming Tran Advisor: Mrs. Rachel Sandler Assistant: Tina Varghese ABSTRACT Camellia sinensis, commonly known as green tea, has been shown to possess antimicrobial properties and to lower the risk of cardiovascular disease and periodontal diseases. This study investigates the effects of brewing green tea at varying concentrations and durations on its antimicrobial activity against common oral bacteria, such as Streptococcus mutans, Porphyromonas gingivalis, and Staphylococcus epidermis. Gram stain tests revealed that our bacteria cultures had a mixture of Gram-positive and Gram-negative bacteria. A paper disk diffusion test revealed that increasing the concentration of green tea and decreasing brewing time increased the zones of inhibition; the tea brewed at a concentration of 80 mg/mL for 20 minutes had the greatest antibacterial effect. In the mouthwash paper disk diffusion test, a new bottle of Scope® was found to be most effective against common oral bacteria, while Listerine® was found to have little effect. The minimum inhibitory concentration test implied a positive correlation between the concentration of green tea and bacterial growth. Tests indicated that Scope® had a considerable effect against bacterial growth, green tea had minimal effect, and water had no effect; however, these results were inconclusive due to small sample size. As confirmed by the study, green tea does have antibacterial properties, but further investigations are required to make a definitive conclusion. INTRODUCTION Green tea is a beverage made from the evergreen plant Camellia sinensis and has been enjoyed for thousands of years. Generally, Camellia sinensis leaves are added to hot water and allowed to brew for several minutes. The resulting beverage lowers the risk for cardiovascular disease and periodontal diseases, and it also has antibacterial and antimicrobial properties1. However, many of its health benefits are not well known since studies on green tea have been conducted only fairly recently. Green tea has long been believed to be beneficial to one’s health and has a long history of widespread consumption. Evidence shows that green tea was consumed as early as the third century AD, yet multiple stories suggest it was brewed much earlier. One legend says that in 2737 B.C. an herbalist named Shen Nung was boiling water to drink while resting under a tree. A breeze caused green tea leaves to fall into his steaming water. When he drank the resulting liquid, Shen Nung was pleasantly surprised by the stimulating taste, and the tradition of tea consumption began. Since the third century, green tea has been used for medicinal purposes, such as depression, stomach problems, and anxiety. The cultivation of green tea rapidly increased during the Tang Dynasty2, and Lu Yu wrote a famous book called Tea Classic, which [3-1] discussed the production, consumption, and culture of green tea3. Around 1211, a Buddhist name Eisai wrote Kissa Yohjoh Ki, the first book discussing the health benefits of green tea on the “five vital organs4.” During the Ming Dynasty in China, green tea became a common drink of the Chinese populace and helped to prevent scurvy in Chinese seamen due to its vitamin C concentration. Today, China and Japan are the world’s leading producers of green tea2, the second most popular drink in the world after water3. Publicized studies on the health benefits of green tea have only been available since the 1990’s. However, green tea’s popularity in the West can be attributed to the growing interest in its potential health benefits2. Green tea’s numerous health benefits are the result of the large percentage of polyphenols found within the tea, even though the polyphenol content varies due to environmental factors like rainfall and season. The major polyphenols are the catechins: epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), epigallate (EG), and catechin (C). EGCG, the most active component of green tea, provides most of its properties because it contains multiple chemically reactive hydroxyl groups (Fig. 1). In one study where green tea leaves were kept in the mouth for several minutes, high concentrations of catechins remained in the mouth after rinsing. These results suggest that tea leaves are a dependable natural source of catechins8. Green tea also contains a myriad of other compounds including gallic acid, quercetin, kaempferol, myricetin, caffeic acid, and chlorogenic acid1. In addition to green tea, black and oolong tea are also common beverages derived from Camellia sinensis that contain polyphenols. However, black tea is fully fermented, while oolong tea is partially fermented. The fermentation process oxidizes many of polyphenols catalyzed by polyphenol oxidase, degrading EGCG and reducing tea’s antibacterial potency. Unlike black and oolong tea, green tea is unfermented, thus containing the highest concentrations of polyphenols and most likely possessing the greatest antibacterial effect1. Fig. 1. Chemical structures of major catechins1. The most common polyphenols in green tea. [3-2] Many conditions, including high cholesterol, diabetes, liver and kidney complications, aging and related degenerative diseases, can be traced back to free radicals, which are extremely reactive in the body. The polyphenols in green tea, mainly EGCG, display anti-oxidative properties by acting as free radical scavengers. The flavonoids can react with free radicals and effectively eliminate possible negative health effects. The presence of three hydroxyl moieties at 3’, 4’, and 5’ on the B ring in the EGCG are primarily responsible for this inhibitory activity5. Previous studies have found that green tea polyphenols can decrease LDL cholesterol levels, thereby increasing the ratio of good cholesterol (HDL) to bad cholesterol (LDL)1. In addition, EGCG has been found to lower plasma cholesterol and triglyceride levels, consequently reducing the risk of cardiovascular disease and inhibiting the growth of abnormal blood clots6. Several studies testing green tea’s anticarcinogenic effects have concluded that green tea prevents and inhibits several forms of cancer, including biliary tract, bladder, breast, colon, esophageal, prostate, and skin cancers. Cancer is the uncontrolled growth and division of cells, which interferes with normal body processes. Cancer cells rely on inflammation to promote tumor angiogenesis. The polyphenols reduce this form of inflammation, effectively inhibiting cancer progression7. In addition, the polyphenols in green tea prevent the growth of blood vessels, slowing down metastasis by inhibiting the distribution of nutrients to the cancer cells6. Green tea also slows the release of tumor necrosis kappa-B function, which is critical for tumor growth (Fig. 2). Furthermore, EGCG and other polyphenols found in green tea promote cell cycle arrest and induce apoptosis in cancerous cells. The polyphenols, which contain strong nucleophilic centers, react with the electrophilic carcinogenic species in order to prevent tumorigenesis. In addition to activating killer caspases, this reaction changes the expression of cell cycle proteins, specifically the Bax/Bc12 function. Moreover, polyphenols can arrest tumor growth internally because EGCG regulates signal transduction pathways involved in cell proliferation, transformation, and metastasis. These polyphenols affect the cancer cells without harming normal body cells, making green tea viable for cancer research. Although the anticarcinogenic effects of green tea have been widely observed, much research has yet to be done in order to determine specific mechanisms that inhibit cancer growth7. Fig. 2: Anticarcinogenic property of green tea1. Various effects of green tea polyphenols in the body that either directly or indirectly inhibit cancer. [3-3] In addition to the health benefits of the polyphenols, these compounds also exhibit powerful antimicrobial and antiviral properties. For instance, green tea actively strengthens the immune system by preventing the binding of HIV to human T-cells. Evidence shows that the EGCG inhibits the infection of human CD4 lymphocytes by HIV. However, further research is required to fully understand green tea’s antiviral effects1. Many studies have shown EGCG to be the most effective antibacterial polyphenol at typical or slightly lower concentrations than found in regular brewed green tea8. The exact mechanisms of EGCG’s antibacterial activity are unknown, but it is believed that EGCG disrupts the cell membrane and prevents DNA supercoiling, ultimately leading to the destruction of the bacterial cell. In vitro experiments suggest that EGCG affects fungal pathogens, Gram-positive bacteria, and Gram-negative bacteria, but Gram-positive bacteria are particularly vulnerable to the polyphenols. The difference between Gram-positive and Gram-negative bacteria lies in the cell wall. The Gram-positive cell wall has several layers of peptidoglycans that are joined together to form a thick, rigid wall, whereas the Gram-negative cell wall has an additional membrane covering the thinner wall of peptidoglycans. This outer membrane contains lipopolysaccharides and lipoproteins, which are vital to the bacteria’s survival under enormous bacterial pressure (Fig. 3)9. Fig. 3: Bacterial
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